Phase intensity nanoscope (PINE) opens long-time investigation windows of living matter

Fundamental to all living organisms and living soft matter are emergent processes in which the reorganization of individual constituents at the nanoscale drives group-level movements and shape changes at the macroscale over time. However, light-induced degradation of fluorophores, photobleaching, is a significant problem in extended bioimaging in life science. Here, we report opening a long-time investigation window by nonbleaching phase intensity nanoscope: PINE. We accomplish phase-intensity separation such that nanoprobe distributions are distinguished by an integrated phase-intensity multilayer thin film (polyvinyl alcohol/liquid crystal). We overcame a physical limit to resolve sub-10 nm cellular architectures, and achieve the first dynamic imaging of nanoscopic reorganization over 250 h using PINE. We discover nanoscopic rearrangements synchronized with the emergence of group-level movements and shape changes at the macroscale according to a set of interaction rules with importance in cellular and soft matter reorganization, self-organization, and pattern formation.


Nanoprobe Synthesis
In order for nanoscopic imaging, scaling up to distributions of nanoprobes as well as structurally stabilized nanoprobes are required. To achieve 100% CTA+ free necessary for structural stabilization, gold nanorods 59 nm  20 nm were synthesized by a bromide-free seed-mediated growth followed by an adaptation of round-trip phase transfer to achieve CTA+ free and passivate surfaces with 90% SH-PEG-COOH and 10% mPEG-SH 1 . To crosslink, EDC/NHS bioconjugation chemistry was employed to form NH2− groups on the nanoprobes. Activated nanoprobes (OD=15, 500 µL) and actin antibody (200 μg/mL) were crosslinked by mixing at a 1:1 ratio for 90 minutes while gentle rotating. After crosslinking, anti-actin-nanoprobes were washed 3 times by centrifugation. After the final wash, supernatant was decanted and anti-actin-nanoprobes were resuspended in 500 µL PBS. To verify CTA+ free, elemental compositions of samples were analyzed by X-ray photoelectron spectroscopy (XPS) analysis.

Fixed Cells
Cells were cultured in media supplemented with 10% FBS and maintained in a 37°C incubator with 5% CO2 humidified air. Cells were cultured to 70% confluency on fibronectin-coated glass coverslips. Blocking with 3% BSA in culture medium was then performed for 30 minutes followed by washing 3 times with PBS and incubated in culture medium. Cells were incubated with antiactin-nanorods (OD=15, 75 µL) in culture medium for six hours followed by 3 times washing with PBS. Cells were treated with 4% paraformaldehyde and then incubated with DAPI for 10 minutes, followed by washing 3 times.

Live Cells
Cells were cultured in media supplemented with 10% FBS and maintained in a 37°C incubator with 5% CO2 humidified air. Cells were cultured to 70% confluency on fibronectin-coated glass coverslips in culture medium with 10% FBS at 37°C. To synchronize cells, cells were incubated in culture medium with 0% FBS for 24 hours at 37°C. To re-enter the cell cycle, cells were incubated in culture medium with 20% FBS for 30 hours at 37°C and then returned back to culture medium with 10% FBS. Blocking with 3% BSA in culture medium was then performed for 30 minutes followed by washing 3 times with PBS and incubated in culture medium. Cells were incubated with anti-actin-nanorods (OD=15, 75 µL) in culture medium for six hours followed by washing 3 times with PBS. Cells on coverslips were then assembled into a chamber on a temperature-controlled microscope stage at 37°C. Culture medium was perfused into the chamber at 0.5 mL/min at 37°C with 5% CO2. Cells were cultured and imaged for 2 days to follow dividing cells and cells which did not divide.

In Vitro Actin
Unlabeled G-actin from rabbit skeletal muscle (1 mg/mL) was prepared in storage buffer (5 mM Tris-HCl pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM TCEP). To crosslink, EDC/NHS bioconjugation chemistry was employed to form NH2− groups on the nanoprobes. Activated nanoprobes (OD=15, 100 µL) were added to the actin solution at a 1:1 ratio and incubated for 1 hr. After the crosslinking step, the sample was gently washed 3 times with DI water. For the fluorescent imaging, unlabeled G-actin was added to fluoroscein labeled G-actin at a 1:1 ratio in storage buffer (5 mM Tris-HCl pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM TCEP). Labeled actin was flowed into a chamber coated with 1% methylcellulose and imaged for 1.5 weeks.

XPS
To characterize material composition, samples were deposited on clean silicon substrates for Xray photoelectron spectroscopy (XPS) analysis. Spectra were acquired using monochromatic Al X-rays at 14 kV and 12 mA, 1 eV scan step size, 60 s sweep time with analyzer at 160 eV pass energy, averaged by two times of sweeping to remove the noise, and calibrated with Au 4f7/2 at 84.00 eV.

Electron Beam Lithography
Gold nanorods 125 nm  44 nm were fabricated using electron beam lithography on a glass substrate. A 100 nm thick 950k A2 PMMA photoresist was first spin-coated onto a glass slide, followed by Ni beads drop-cast on the edge of the substrate to aid focusing in the e-beam process.
Gold nanorods were then patterned by electron beam lithography (Jeol 6300FS) using a beam size of 1.8 nm and a step size of 0.125 nm to ensure uniform patterning while preserving the designed shape. After developing the patterns, a layer of chromium (20 Å) and a layer of gold (300 Å) were deposited by ebeam evaporation (Enerjet). A lift-off process was subsequently performed using acetone to dissolve the photoresist.
In this work, we designed an integrated phase-intensity multilayer thin film (herein referred to as PI) to be displacement-free. Distinctly different from our previously reported work constructed with standard waveplate stacks 3 , here the compact multilayer design was integrated into the infinity space between the objective and tube lens, enabling high transmission for nanoscopy. The optical axis of PI was stationary such that the center coordinates during modulation (x, y) were equal to the center coordinates prior to modulation (x0, y0), enabling zero displacement for precise nanoscopy (Eq. 3). For validation of displacement-free PI, the scattered electric field from a single nanorod was reshaped by PI as = + . Using PI, we modulated  from 0 to 2 which varies the phase difference between Ex and Ey from -   according to Eq. 1. After phase to intensity conversion by PI, we observed the nanorod remained precisely at the same position in the imaging plane over the modulation range following Eq. 3 using PI ( Figure S11). As a measure of displacement, we quantified the root mean square deviation of the images acquired over the modulation range. The normalized root-mean-square deviation of the images was calculated for each pixel, where the experimental value of the intensity was compared with the theoretical value of the intensity fitted to sinusoidal functions over the modulation range.  Figure S17c).

Supplementary Note III: Validation of temporal capabilities
We validated the response time of the PI to determine the timescale of dynamic activities that can be captured. To validate long timescale temporal capabilities, we followed the temporal evolution of cellular architectures (actin) over time scales of weeks (Figure 4c). We observed local rearrangements below the diffraction limit ( Figure S22) in agreement with theoretical modeling ( Figure S23). No local rearrangements in the negative control ( Figure S24).